Project 2 - Abstract One of the most challenging problems in HIV-1 vaccine research has been the induction of broad and long-lasting protective antibody (Ab) responses that include Ab with both neutralizing and antibody dependent cellular cytotoxic (ADCC) activities in systemic and mucosal compartments. Our leading hypothesis is that the immunization with polyvalent DNA and recombinant proteins will provide the immune system with diverse spectrum of HIV-1 Envs that will drive the development of Ab responses with a greater breadth. This novel approach could shorten the time required to develop broad Ab responses and may induce BNAb and ADCC Ab responses directed to multiple HIV-1 Env epitopes. Moreover, a practical and effective vaccine must also elicit long-lived Ab responses in order to provide durable protection. Thus, our secondary hypothesis is that the longevity of the B cell response is correlated with the priming and expansion of T follicular helper (Tfh) cells by the vaccine. Lastly, we hypothesize that the degree of somatic mutation of Env-specific Ab responses elicited by this immunization regime will correlate with the breadth of functional Ab responses but that the vaccine will not elicit the levels of Ab mutation observed in the course of natural infection. We will isolate monoclonal Abs (mAbs) with reflective of the vaccine-induced plasma activity and determine the somatic mutation and gene usage of these mAbs from those immunized animals that develop the highest degree of Ab breadth. We will further define the maturational pathways of these mAbs using Ab gene deep sequencing techniques to determine how the vaccine studied in this project compares with previously characterized human vaccine studies. In order to address our hypothesis, we have defined 3 AIMs for our project: AIM.1 To analyze the breadth of vaccine-induced binding, neutralizing, and ADCC Ab responses; AIM. 2 To analyze the longevity of vaccine-induced Ab responses and correlate with the presence of Tfh responses; AIM. 3 To determine the somatic mutation and gene usage of these mAbs from those immunized animals that develop the highest degree of Ab breadth.